PEG-urate oxidase conjugates and use thereof

ABSTRACT

A naturally occurring or recombinant urate oxidase (uricase) covalently coupled to poly(ethylene glycol) or poly(ethylene oxide) (both referred to as PEG), wherein an average of 2 to 10 strands of PEG are conjugated to each uricase subunit and the PEG has an average molecular weight between about 5 kDa and 100 kDa. The resulting PEG-uricase conjugates are substantially non-immunogenic and retain at least 75% of the uricolytic activity of the unmodified enzyme.

STATEMENT OF GOVERNMENT RIGHTS

[0001] A portion of the research described in this application was madewith support from Grant DK48529 from the National Institutes of Health.Accordingly, the U.S. government may have certain rights in thisinvention.

FIELD OF THE INVENTION

[0002] The present invention relates to chemical modification ofproteins to prolong their circulating lifetimes and reduce theirimmunogenicity. More specifically, the invention relates to conjugationof poly(ethylene glycols) or poly(ethylene oxides) to urate oxidases,which substantially eliminates urate oxidase immunogenicity withoutcompromising its uricolytic activity.

BACKGROUND OF THE INVENTION

[0003] Statements contained in this background section do not constitutean admission of prior art, but instead reflect the inventors' ownsubjective comments on and interpretations of the state of the art atthe time the invention was made. These interpretations may includepersonal, heretofore undisclosed, insights of the inventors, whichinsights were not themselves part of the prior art.

[0004] Urate oxidases (uricases; E.C. 1.7.3.3) are enzymes that catalyzethe oxidation of uric acid to a more soluble product, allantoin, apurine metabolite that is more readily excreted. Humans do not produceenzymatically active uricase, as a result of several mutations in thegene for uricase acquired during the evolution of higher primates. Wu,X, et al., (1992) J Mol Evol 34:78-84. As a consequence, in susceptibleindividuals, excessive concentrations of uric acid in the blood(hyperuricemia) and in the urine (hyperuricosuria) can lead to painfularthritis (gout), disfiguring urate deposits (tophi) and renal failure.In some affected individuals, available drugs such as allopurinol (aninhibitor of uric acid synthesis) produce treatment-limiting adverseeffects or do not relieve these conditions adequately. Hande, K R, etal., (1984) Am J Med 76:47-56; Fam, A G, (1990) Bailliére's ClinRheumatol 4:177-192. Injections of uricase can decrease hyperuricemiaand hyperuricosuria, at least transiently. Since uricase is a foreignprotein in humans, however, even the first injection of the unmodifiedprotein from Aspergillus flavus has induced anaphylactic reactions inseveral percent of treated patients (Pui, C-H, et al., (1997) Leukemia11: 1813-1816), and immunologic responses limit its utility for chronicor intermittent treatment. Donadio, D, et al., (1981) Nouv Presse Méd10:711-712; Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic50:553-554.

[0005] The sub-optimal performance of available treatments forhyperuricemia has been recognized for several decades. Kissel, P, etal., (1968) Nature 217:72-74. Similarly, the possibility that certaingroups of patients with severe gout might benefit from a safe andeffective form of injectable uricase has been recognized for many years.Davis, F F, et al., (1978) in G B Broun, et al., (Eds.) EnzymeEngineering, Vol. 4 (pp. 169-173) New York, Plenum Press; Nishimura, H,et al., (1979) Enzyme 24:261-264; Nishimura, H, et al., (1981) Enzyme26:49-53; Davis, S, et al., (1981) Lancet 2(8241):281-283; Abuchowski,A, et al., (1981) J Pharmacol Exp Ther 219:352-354; Chen, R H-L, et al.,(1981) Biochim Biophys Acta 660:293-298; Chua, C C, et al., (1988) AnnInt Med 109:114-117; Greenberg, M L, et al., (1989) Anal Biochem176:290-293. Uricases derived from animal organs are nearly insoluble insolvents that are compatible with safe administration by injection. U.S.Pat. No. 3,616,231. Certain uricases derived from plants or frommicroorganisms are more soluble in medically acceptable solvents.However, injection of the microbial enzymes quickly inducesimmunological responses that can lead to life-threatening allergicreactions or to inactivation and/or accelerated clearance of the uricasefrom the circulation. Donadio, et al., (1981); Leaustic, et al., (1983).Enzymes based on the deduced amino acid sequences of uricases frommammals, including pig and baboon, or from insects, such as, forexample, Drosophila melanogaster or Drosophila pseudoobscura (Wallrath,L L, et al., (1990) Mol Cell Biol 10:5114-5127), have not been suitablecandidates for clinical use, due to problems of immunogenicity andinsolubility at physiological pH.

[0006] Covalent modification of proteins with poly(ethylene glycol) orpoly(ethylene oxide) (both referred to as PEG), has been used toincrease protein half-life and reduce immunogenicity. U.S. Pat. Nos.4,179,337, 4,766,106, and 4,847,325; Saifer, M G P, et al., (1994) AdvExp Med Biol 366:377-387. The coupling of PEG of high molecular weightto produce conjugates with prolonged circulating lifetimes and/ordecreased immunogenicity, while conserving functional activity, waspreviously demonstrated for another enzyme, superoxide dismutase(Somack, R, et al., (1991) Free Rad Res Commun 12-13:553-562; U.S. Pat.Nos. 5,283,317 and 5,468,478) and for other types of proteins, e.g.,cytokines (Saifer, M G P, et al., (1997) Polym Preprints 38:576-577;Sherman, M R, et al., (1997) in J M Harris, et al., (Eds.),Poly(ethylene glycol) Chemistry and Biological Applications. ACSSymposium Series 680 (pp. 155-169) Washington, D.C.: American ChemicalSociety). Conjugates of uricase with polymers other than PEG have alsobeen described. U.S. Pat. No. 4,460,683.

[0007] In nearly all of the reported attempts to PEGylate uricase (i.e.to covalently couple PEG to uricase), the PEG was attached primarily toamino groups, including the amino-terminal residue and the availablelysine residues. In the uricases commonly used, the total number oflysines in each of the four identical subunits is between 25(Aspergillus flavus (U.S. Pat. No. 5,382,518)) and 29 (pig (Wu, X, etal., (1989) Proc Natl Acad Sci USA 86:9412-9416)). Some of the lysinesare unavailable for PEGylation in the native conformation of the enzyme.The most common approach to reducing the immunogenicity of uricase hasbeen to couple large numbers of strands of low molecular weight PEG.This has invariably resulted in large decreases in the enzymaticactivity of the resultant conjugates.

[0008] Previous investigators have used injected uricase to catalyze theconversion of uric acid to allantoin in vivo. See Pui, et al., (1997).This is the basis for the use in France and Italy of uricase from thefungus Aspergillus flavus (Uricozyme®) to prevent or temporarily correctthe hyperuricemia associated with cytotoxic therapy for hematologicmalignancies and to transiently reduce severe hyperuricemia in patientswith gout. Potaux, L, et al., (1975) Nouv Presse Méd 4:1109-1112;Legoux, R, et al., (1992) J Biol Chem 267:8565-8570; U.S. Pat. Nos.5,382,518 and 5,541,098. Because of its short circulating lifetime,Uricozyme® requires daily injections. Furthermore, it is not well suitedfor long-term therapy because of its immunogenicity.

[0009] A single intravenous injection of a preparation of Candida utilisuricase coupled to 5 kDa PEG reduced serum urate to undetectable levelsin five human subjects whose average pre-injection serum urateconcentration was 6.2 mg/dL, which is within the normal range. Davis, etal., (1981). The subjects were given an additional injection four weekslater, but their responses were not reported. No antibodies to uricasewere detected following the second (and last) injection, using arelatively insensitive gel diffusion assay. This reference reported noresults from chronic or subchronic treatments of human patients orexperimental animals.

[0010] A preparation of uricase from Arthrobacter protoformiae coupledto 5 kDa PEG was used to temporarily control hyperuricemia in a singlepatient with lymphoma whose pre-injection serum urate concentration was15 mg/dL. Chua, et al., (1988). Because of the critical condition of thepatient and the short duration of treatment (four injections during 14days), it was not possible to evaluate the long-term efficacy or safetyof the conjugate.

[0011] In this application, the term “immunogenicity” refers to theinduction of an immune response by an injected preparation ofPEG-modified or unmodified uricase (the antigen), while “antigenicity”refers to the reaction of an antigen with preexisting antibodies.Collectively, antigenicity and immunogenicity are referred to as“immunoreactivity.” In previous studies of PEG-uricase, immunoreactivitywas assessed by a variety of methods, including: 1) the reaction invitro of PEG-uricase with preformed antibodies; 2) measurements ofinduced antibody synthesis; and 3) accelerated clearance rates afterrepeated injections.

[0012] Previous attempts to eliminate the immunogenicity of uricasesfrom several sources by coupling various numbers of strands of PEGthrough various linkers have met with limited success. PEG-uricases werefirst disclosed by F F Davis and by Y Inada and their colleagues. Davis,et al., (1978); U.S. Pat. No. 4,179,337; Nishimura, et al., (1979);Japanese Patents 55-99189 and 62-55079. The conjugate disclosed in the'337 patent was synthesized by reacting uricase of unspecified originwith a 2,000-fold molar excess of 750 dalton PEG, indicating that alarge number of polymer molecules was likely to have been attached toeach uricase subunit. The '337 patent discloses the coupling of eitherPEG or poly(propylene glycol) with molecular weights of 500 to 20,000daltons, preferably about 500 to 5,000 daltons, to provide active,water-soluble, non-immunogenic conjugates of various polypeptidehormones and enzymes including oxidoreductases, of which uricase is oneof three examples. In addition, the '337 patent emphasizes the couplingof 10 to 100 polymer strands per molecule of enzyme, and the retentionof at least 40% of enzymatic activity. No test results were reported forthe extent of coupling of PEG to the available amino groups of uricase,the residual specific uricolytic activity, or the immunoreactivity ofthe conjugate.

[0013] Data from 13 citations relating to PEGylation of uricase aresummarized in Table 1. Some of these results are also presentedgraphically in FIGS. 1A-2B. Seven of these publications describesignificant decreases in uricolytic activity measured in vitro caused bycoupling various numbers of strands of PEG to uricase from Candidautilis. Coupling a large number of strands of 5 kDa PEG to porcine liveruricase gave similar results, as described in both the Chen publicationand a symposium report by the same group. Chen, et al., (1981); Davis,et al., (1978).

[0014] Among the studies summarized in Table 1, the immunoreactivity ofuricase was reported to be decreased by PEGylation in seven of them andeliminated in five of them. In three of the latter five studies, theelimination of immunoreactivity was associated with profound decreasesin uricolytic activity—to at most 15%, 28%, or 45% of the initialactivity. Nishimura, et al., (1979) (15% activity); Chen, et al., (1981)(28% activity); Nishimura, et al., (1981) (45% activity). In the fourthreport, PEG was reported to be coupled to 61% of the available lysineresidues, but the residual specific activity was not stated. Abuchowski,et al., (1981). However, a research team that included two of the samescientists and used the same methods reported elsewhere that this extentof coupling left residual activity of only 23-28%. Chen, et al., (1981).The 1981 publications of Abuchowski et al., and Chen et al., indicatethat to reduce the immunogenicity of uricase substantially, PEG must becoupled to approximately 60% of the available lysine residues (Table 1).The fifth publication in which the immunoreactivity of uricase wasreported to have been eliminated does not disclose the extent of PEGcoupling, the residual uricolytic activity, or the nature of thePEG-protein linkage. Veronese, F M, et al., (1997) in J M Harris, etal., (Eds.), Poly(ethylene glycol) Chemistry) and BiologicalApplications. ACS Symposium Series 680 (pp. 182-192) Washington, D.C.:American Chemical Society.

[0015] Conjugation of PEG to a smaller fraction of the lysine residuesin uricase reduced but did not eliminate its immunoreactivity inexperimental animals. Tsuji, J, et al., (1985) Int J Immunopharmacol7:725-730 (28-45% of the amino groups coupled); Yasuda, Y, et al.,(1990) Chem Pharm Bull 38:2053-2056 (38% of the amino groups coupled).The residual uricolytic activities of the corresponding adducts rangedfrom <33% (Tsuji, et al.) to 60% (Yasuda, et al.) of their initialvalues. Tsuji, et al., synthesized PEG-uricase conjugates with 7.5 kDaand 10 kDa PEGs, in addition to kDa PEG. All of the resultant conjugateswere somewhat immunogenic and antigenic, while displaying markedlyreduced enzymatic activities (Table 1; FIGS. 1A-1B).

[0016] A PEGylated preparation of uricase from Candida utilis that wassafely administered twice to each of five humans was reported to haveretained only 11% of its initial activity. Davis, et al., (1981).Several years later, PEG-modified uricase from Arthrobacter protoformiaewas administered four times to one patient with advanced lymphoma andsevere hyperuricemia. Chua, et al., (1988). While the residual activityof that enzyme preparation was not measured, Chua, et al., demonstratedthe absence of anti-uricase antibodies in the patient's serum 26 daysafter the first PEG-uricase injection, using an enzyme-linkedimmunosorbent assay (ELISA).

[0017] As summarized in Table 1, previous studies of PEGylated uricaseshow that catalytic activity is markedly depressed by coupling asufficient number of strands of PEG to decrease its immunoreactivitysubstantially. Furthermore, most previous preparations of PEG-uricasewere synthesized using PEG activated with cyanuric chloride, a triazinederivative (2,4,6-trichloro-1,3,5-triazine) that has been shown tointroduce new antigenic determinants and to induce the formation ofantibodies in rabbits. Tsuji, et al., (1985).

[0018] Japanese Patent 3-148298 to A Sano, et al., discloses modifiedproteins, including uricase, derivatized with PEG having a molecularweight of 1-12 kDa that show reduced antigenicity and “improvedprolonged” action, and methods of making such derivatized peptides.However, there are no disclosures regarding strand counts, enzymeassays, biological tests or the meaning of “improved prolonged.”Japanese Patents 55-99189 and 62-55079, both to Y Inada, discloseuricase conjugates prepared with PEG-triazine or bis-PEG-triazine(denoted as PEG₂ in Table 1), respectively. See Nishimura, et al., (1979and 1981). In the first type of conjugate, the molecular weights of thePEGs were 2 kDa and 5 kDa, while in the second, only 5 kDa PEG was used.Nishimura, et al., (1979) reported the recovery of 15% of the uricolyticactivity after modification of 43% of the available lysines with linear5 kDa PEG, while Nishimura, et al., (1981) reported the recovery of 31%or 45% of the uricolytic activity after modification of 46% or 36% ofthe lysines, respectively, with PEG₂.

SUMMARY OF THE INVENTION

[0019] Previous studies teach that when a significant reduction in theimmunogenicity and/or antigenicity of uricase is achieved by PEGylation,it is invariably associated with a substantial loss of uricolyticactivity. The safety, convenience and cost-effectiveness ofbiopharmaceuticals are all adversely impacted by decreases in theirpotencies and the resultant need to increase the administered dose.Thus, there is a need for a safe and effective alternative means forlowering elevated levels of uric acid in body fluids, including bloodand urine. The present invention provides a substantiallynon-immunogenic PEG-uricase that retains all or nearly all of theuricolytic activity of the unmodified enzyme.

[0020] One embodiment of the present invention is a conjugate of urateoxidase (uricase) that retains at least about 75% of the uricolyticactivity of unconjugated uricase and has substantially reducedimmunogenicity. This embodiment includes a purified uncase in which eachsubunit may be covalently linked to an average of 2 to 10 strands ofPEG, which may be linear or branched, wherein each molecule of PEG mayhave a molecular weight between about 5 kDa and 100 kDa. The uricase ofthis aspect of the invention may be recombinant. Whether recombinant ornot, the uricase may be of mammalian origin. In one aspect of thisembodiment, the uricase may be porcine, bovine or ovine liver uricase.In another aspect of this embodiment, the uricase may be chimeric. Thechimeric uricase may contain portions of porcine liver and/or baboonliver uricase. For example, the chimeric uricase may be pig-baboonchimeric uricase (PBC uricase) or porcine uricase containing themutations R291K and T301S (PKS uricase) (see sequences in FIG. 6 andresults of physiological and immunological studies in FIGS. 7-12).Alternatively, the uricase may be baboon liver uricase in which tyrosine97 has been replaced by histidine, whereby the specific activity of theuricase may be increased by at least about 60%. The uricase of theinvention, whatever the origin, may also be in a form that is truncated,either at the amino terminal, or at the carboxyl terminal, or at bothterminals. Likewise, the uricase may be fungal or microbial uricase. Inone aspect of this embodiment, the fungal or microbial uricase may be anaturally occurring or recombinant form of uricase from Aspergillusflavus, Arthrobacter globiformis or Candida utilis. Alternatively, theuricase may be an invertebrate uricase, such as, for example, anaturally occurring or recombinant form of uncase from Drosophilamelanogaster or Drosophila pseudoobscura. The uricase of the inventionmay also be a plant uricase, for example, a naturally occurring orrecombinant form of uricase from soybean root nodule (Glycine max). ThePEG may have an average molecular weight between about 5 kDa and 100kDa; preferably the PEG may have an average molecular weight betweenabout 10 kDa and 60 kDa; more preferably, the PEG may have an averagemolecular weight between about 20 kDa and about 40 kDa, such as, forexample, 30 kDa. The average number of covalently coupled strands of PEGmay be 2 to 10 strands per uricase subunit; preferably, the averagenumber of covalently coupled strands may be 3 to 8 per subunit; morepreferably, the average number of strands of PEG may be 4 to 6 persubunit. In one aspect of this embodiment, the uricase may betetrameric. The strands of PEG may be covalently linked to uricase viaurethane (carbamate) linkages, secondary amine linkages, and/or amidelinkages. When the uricase is a recombinant form of any of the uricasesmentioned herein, the recombinant form may have substantially thesequence of the naturally occurring form.

[0021] Another embodiment of the present invention is a pharmaceuticalcomposition for lowering uric acid levels in body fluids, containing anyof the PEG-uricase conjugates described above and a pharmaceuticallyacceptable carrier. The composition may be stabilized by lyophilizationand also may dissolve promptly upon reconstitution to provide solutionssuitable for parenteral administration.

[0022] The present invention also provides a method for lowering uricacid levels in body fluids and tissues of a mammal. The method includesadministering to a mammal an effective uric acid-lowering amount ofPEG-uricase. The PEG-uricase may be a purified uricase of two or moresubunits in which each subunit may be covalently linked to an average of2 to 10 strands of linear or branched PEG, wherein each molecule of PEGmay have a molecular weight between about 5 kDa and 100 kDa, in apharmaceutically acceptable carrier. The mammal may be a human. Theadministering step may be, for example, injection by intravenous,intradermal, subcutaneous, intramuscular or intraperitoneal routes orinhalation of an aerosolized preparation. The elevated uric acid levelsmay be in blood, urine and/or other body fluids and tissues, and may beassociated with gout, tophi, renal insufficiency, organ transplantationor malignant disease.

[0023] Other embodiments of the present invention are a method forisolating a tetrameric form of uricase from a solution containingmultiple forms of uricase and the product of that method. Initially, thesolution may contain tetrameric uricase and uricase aggregates. Themethod may include the steps of: applying the solution to at least oneseparation column at a pH between about 9 and 10.5, such as, forexample, 10.2; recovering fractions of the eluate and identifying thosethat may contain isolated tetrameric uricase, wherein the fractions aresubstantially free of uricase aggregates; and pooling the fractions ofthe isolated tetrameric uricase. The separation column may be based onion exchange, size exclusion, or any other effective separationproperty. The method may also include analysis of the fractions todetermine the presence of tetrameric uricase and/or the absence ofuricase aggregates. For example, such analysis may include highperformance liquid chromatography (HPLC), other chromatographic methods,light scattering, centrifugation and/or electrophoresis. In one aspectof this embodiment, the purified tetrameric uricase may contain lessthan about 10% uricase aggregates.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1A shows the retention of activity by PEGylated uricase fromCandida utilis as a function of the number of strands of PEG coupled persubunit.

[0025]FIG. 1B shows the retention of activity by PEGylated uricase fromCandida utilis as a function of the total mass of PEG coupled persubunit.

[0026]FIG. 2A shows the retention of activity by PEGylated uricase fromporcine liver as a function of the number of strands of PEG coupled persubunit.

[0027]FIG. 2B shows the retention of activity by PEGylated uricase fromporcine liver as a function of the total mass of PEG coupled persubunit.

[0028]FIG. 3A shows the retention of activity by PEGylated pig-baboonchimeric (PBC) uricase as a function of the number of strands coupledper subunit.

[0029]FIG. 3B shows the retention of activity by PEGylated PBC uricaseas a function of the total mass of PEG coupled per subunit.

[0030]FIG. 4A shows the retention of activity by PEGylated uricase fromAspergillus flavus as a function of the number of strands of PEG coupledper subunit.

[0031]FIG. 4B shows the retention of activity by PEGylated uricase fromAspergillus flavus as a function of the total mass of PEG coupled persubunit.

[0032]FIG. 5A shows the retention of activity by PEGylated recombinantsoybean root nodule uricase as a function of the number of strands ofPEG coupled per subunit.

[0033]FIG. 5B shows the retention of activity by PEGylated recombinantsoybean root nodule uricase as a function of the total mass of PEGcoupled per subunit.

[0034]FIG. 6 shows the deduced amino acid sequences of pig-baboonchimeric uricase (PBC uricase), PBC uricase that is truncated at boththe amino and carboxyl terminals (PBC-NT-CT) and porcine uricasecontaining the mutations R291K and T301 S (PKS uricase), compared withthe porcine and baboon sequences.

[0035]FIG. 7 shows the activity of uricase in mouse serum 24 h aftereach of four or five intraperitoneal injections of PEG-modified PBCuricase, relative to the value 24 h after the first injection.

[0036]FIG. 8 shows the inverse relationship between the activity ofinjected PEG-modified PBC uricase in the serum of a uricase-deficientmouse and the concentrations of uric acid in the serum and urine.

[0037]FIG. 9 shows the decreased severity of a urine-concentratingdefect in uricase-deficient (uox −/−) mice that were treated withPEG-modified PBC uricase.

[0038]FIG. 10 shows the decreased severity of nephrogenic diabetesinsipidus in uricase-deficient (uox −/−) mice that were treated withPEG-modified PBC uricase.

[0039]FIG. 11 shows the decreased severity of uric acid-inducednephropathy, as visualized by magnetic resonance microscopy, inuricase-deficient (uox −/−) mice that were treated with PEG-modified PBCuricase.

[0040]FIG. 12 shows the accelerated clearance from the circulation ofBALB/c mice of injected PBC uricase octamer, compared with the tetramer,when both were coupled to 5-6 strands of 10 kDa PEG per subunit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention provides improved conjugates ofwater-soluble polymers, preferably poly(ethylene glycols) orpoly(ethylene oxides), with uricases. The invention also providespharmaceutical compositions of the improved conjugates. These conjugatesare substantially non-immunogenic and retain at least 75%, preferably85%, and more preferably 95% or more of the uricolytic activity of theunmodified enzyme. Uricases suitable for conjugation to water-solublepolymers include naturally occurring urate oxidases isolated frombacteria, fungi and the tissues of plants and animals, both vertebratesand invertebrates, as well as recombinant forms of uricase, includingmutated, hybrid, and/or truncated enzymatically active variants ofuricase. Water-soluble polymers suitable for use in the presentinvention include linear and branched poly(ethylene glycols) orpoly(ethylene oxides), all commonly known as PEGs. Examples of branchedPEG are the subject of U.S. Pat. No. 5,643,575. One preferred example oflinear PEG is monomethoxyPEG, of the general structureCH₃O—(CH₂CH₂O)_(n)H, where n varies from about 100 to about 2,300.

[0042] One preferred mammalian uricase is recombinant pig-baboonchimeric uricase, composed of portions of the sequences of pig liver andbaboon liver uricase, both of which were first determined by Wu, et al.,(1989). One example of such a chimeric uricase contains the first 225amino acids from the porcine uricase sequence (SEQ ID NO: 1) and thelast 79 amino acids from the baboon uricase sequence (SEQ ID NO: 2)(pig-baboon uricase, or PBC uricase; see FIG. 6). Another example ofsuch a chimeric uricase contains residues 7-225 of the porcine sequence(SEQ ID NO. 1) and residues 226-301 of the baboon sequence (SEQ ID NO.2); this is equivalent to PBC uricase that is truncated at both theamino and carboxyl terminals (PBC-NT-CT; see FIG. 6). Another example ofsuch a chimeric uricase contains the first 288 amino acids from theporcine sequence (SEQ ID NO: 1) and the last 16 amino acids from thebaboon sequence (SEQ ID NO: 2). Since the latter sequence differs fromthe porcine sequence at only two positions, having a lysine (K) in placeof arginine at residue 291 and a serine (S) in place of threonine atresidue 301, this mutant is referred to as pig-K-S or PKS uricase. PKS,PBC and PBC-NT-CT uricases each have one more lysine residue and, hence,one more potential site of PEGylation than either the porcine or baboonsequence.

[0043] The cDNAs for various mammalian uricases, including PBC uricase,PKS uricase and a recombinant baboon-like uricase, were subcloned andthe optimal conditions were determined for expression in E. coli, usingstandard methods. See Erlich, H A, (Ed.) (1989) PCR Technology.Principles and Applications for DNA Amplification. New York: StocktonPress; Sambrook, J, et al., (1989) Molecular Cloning. A LaboratoryManual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press. The recombinant uricases were extracted, purified andtheir stability and activity were assessed using a modification ofstandard assays. See Fridovich, I, (1965) J Biol Chem 240:2491-2494;Nishimura, et al., (1979), and Example 1.

[0044] In one embodiment of the invention, uricase may be conjugated viaa biologically stable, nontoxic, covalent linkage to a relatively smallnumber of strands of PEG. Such linkages may include urethane (carbamate)linkages, secondary amine linkages, and amide linkages. Variousactivated PEGs suitable for such conjugation are available commerciallyfrom Shearwater Polymers, Huntsville, Ala.

[0045] For example, urethane linkages to uricase may be formed byincubating uricase in the presence of the succinimidyl carbonate (SC) or4-nitrophenyl carbonate (NPC) derivative of PEG. SC-PEG may besynthesized using the procedure described in U.S. Pat. No. 5,612,460,which is hereby incorporated by reference. NPC-PEG may be synthesized byreacting PEG with 4-nitrophenyl chloroformate according to methodsdescribed in Veronese, F M, et al., (1985) Appl Biochem Biotechnol11:141-152, and in U.S. Pat. No. 5,286,637, which is hereby incorporatedby reference. The methods described in the '637 patent are adapted toPEGs of higher molecular weight by adjusting the concentrations of thereactants to maintain similar stoichiometry. An alternative method ofsynthesis of NPC-PEG is described by Büttner, W, et al., East GermanPatent Specification DD 279 486 A1.

[0046] Amide linkages to uricase may be obtained using anN-hydroxysuccinimide ester of a carboxylic acid derivative of PEG(Shearwater Polymers). Secondary amine linkages may be formed using2,2,2-trifluoroethanesulfonyl PEG (tresyl PEG; Shearwater Polymers) orby reductive alkylation using PEG aldehyde (Shearwater Polymers) andsodium cyanoborohydride.

[0047] In conjugates containing PEGs with molecular weights between 5kDa and 30 kDa, the maximum number of strands of PEG that were coupledper subunit, while retaining at least 75% of the uricolytic activity ofthe unmodified enzyme, ranged from an average of 2 strands for soybeanuricase to more than 10 strands for PBC uricase (see assay conditions inExample 1 and results in FIGS. 1A-5B). The latter extent of PEGylationcorresponds to approximately one third of the total amino groups. In oneembodiment of the invention, the average number of strands of PEGcoupled per uricase subunit is between 2 and 10. In a preferredembodiment, the average number of strands of PEG coupled per uricasesubunit is between 3 and 8. In a more preferred embodiment, the averagenumber of covalently linked strands of PEG per uricase subunit isbetween 4 and 6. In another embodiment, the molecular weight of PEG usedfor the coupling reaction is between 5 kDa and 100 kDa, preferablybetween 10 kDa and 60 kDa, and more preferably between 20 kDa and 40kDa, such as, for example 30 kDa.

[0048] There are several factors that may affect the choice of theoptimal molecular weight and number of strands of PEG for coupling to agiven form of uricase. In general, the reduction or elimination ofimmunogenicity without substantial loss of uricolytic activity mayrequire the coupling of relatively more strands of PEG of lowermolecular weight, compared to relatively fewer strands of PEG of highermolecular weight. For example, either 6 strands of 20 kDa PEG persubunit or 4 strands of 30 kDa PEG per subunit might be optimallyeffective. Likewise, each different form of uricase may have a differentoptimum with respect to both the size and number of strands. See FIGS.1A-5B.

[0049] PEG conjugation rendered all of the tested uricases soluble andstable in buffers at physiological pH, without the addition of asubstrate analog or inhibitor, such as 8-azaxanthine that is used as astabilizer in the fungal uricase (Uricozyme®) sold by Sanofi Winthrop inFrance and Italy. Two different conjugates of PBC uricase, onecontaining approximately 6 strands of 10 kDa PEG per subunit and theother containing approximately 2 strands of 19 kDa PEG per subunit,retained significant activity after incubation in mouse serum for morethan one month at 37° C. In addition, several of the conjugates of thisinvention had circulating half-lives in mice that were greater than twodays, in contrast to the approximately 8-hour or 24-hour half-livespreviously reported for PEG-modified mammalian and microbial uricases.Chen, et al., (1981); Fuertges, F, et al., (1990) J Contr Release 11:139-148; Fujita, T, et al., (1991) J Pharmacobiodyn 14:623-629. Longerhalf-lives of injected protein drugs make them more cost-effective andcan lead to improved patient compliance. Prolonged half-life is alsoindicative of products that are better tolerated by the body.

[0050] When PEG conjugates of PBC uricase were prepared from thepurified tetrameric form of the enzyme (four 35 kDa subunits), theydisplayed profoundly reduced immunogenicity in mice (FIG. 7), incontrast to the moderate immunogenicity of PEG conjugates of largerforms of the enzyme (e.g. octamers of the 35 kDa subunit; see FIG. 12),and the very high immunogenicity of the unmodified enzyme. Repeatedinjections of uricase-deficient mice with PEG-uricase of the presentinvention eliminated their hyperuricemia for more than 2 months andprotected the structure and function of their kidneys against uricacid-related damage (FIGS. 8-11).

[0051] Injections of fully active conjugates of PBC uricase with 10 kDaPEG (FIGS. 3A-3B) reduced dramatically the hyperuricemia of homozygous,uricase-deficient mice (FIG. 8). Uric acid levels in the urine were alsoreduced dramatically in all uricase-deficient mice treated withPEG-modified PBC uricase. Uricase-deficient mice received a series ofinjections with a preparation of PEG-uricase similar to that used toobtain the data in FIG. 8. This treatment reduced the severity of aurine-concentrating defect, as demonstrated by measurements of urineosmolality under normal conditions and after a 12-hour period of waterdeprivation (FIG. 9) and by their water consumption and urine output(FIG. 10), compared to the corresponding measurements in untreated,genetically similar mice. It was also demonstrated that ten weeks oftreatment, starting within the first ten days of life, of homozygousuricase-deficient (uox −/−) “knockout” mice with a PEG-uricase of thisinvention decreased the severity of urate-induced disruption of therenal architecture, as visualized by magnetic resonance microscopy (FIG.11). For microscopy methods, see Hedlund, L W, et al., (1991) Fund ApplToxicol 16:787-797; Johnson, G A, et al., (1992) in J C Gore, (Ed.),Reviews of Magnetic Resonance in Medicine, Vol. 4 (pp. 187-219) NewYork: Pergamon Press.

[0052] Purified preparations of naturally occurring and recombinanturicases usually contain a mixture of aggregates of the enzyme, inaddition to the tetrameric (140 kDa) form. The percentage of eachuricase preparation that is in the tetrameric form generally varies fromapproximately 20% to 90%. Despite evidence that unPEGylated aggregatesof several other proteins are highly immunogenic (see, e.g., Moore, W V,et al., (1980) J Clin Endocrinol Metab 51:691-697), previous studies ofPEG-uricase do not describe any efforts to limit the content ofaggregates, suggesting that the potential immunogenicity of thePEG-modified aggregates was not considered. On the basis of theobservations of the present inventors, it appears likely that suchaggregates were present in the enzyme preparations used for previoussyntheses of PEG-uricase. Their presence may have rendered the task ofpreparing non-immunogenic conjugates more difficult. It also appearsthat the large losses of uricolytic activity observed in previousefforts to PEGylate uricase were related to the large number of strandsof low molecular weight PEG that were coupled. On the other hand, themethods of uricase purification and PEGylation described herein permitthe covalent attachment of as many as 10 strands of PEG per subunitwhile retaining more than 75% of the uricolytic activity, at least forcertain uricases, e.g., pig-baboon chimeric uricase and the enzyme fromA. flavus (see FIGS. 3A and 4A).

[0053] In another preferred embodiment, substantially all aggregates ofthe tetrameric form of the enzyme may be removed by ion-exchange orsize-exclusion chromatography at a pH between about 9 and 10.5,preferably 10.2, prior to PEG conjugation of the resulting substantiallytetrameric preparation of uricase. The molecular weight of the unease ineach fraction from the preparative column may be monitored by anysize-dependent analytical technique, including, for example, HPLC,conventional size-exclusion chromatography, centrifugation, lightscattering, capillary electrophoresis or gel electrophoresis in anon-denaturing buffer. For tetrameric unease isolated usingsize-exclusion chromatography, fractions containing only the 140 kDaform of the enzyme may be pooled and used for conjugation to PEG. Fortetrameric uricase isolated using ion-exchange chromatography, fractionsfrom the ion-exchange column may be analyzed with respect to size todetermine which fractions contain substantial amounts of the tetramericform without detectable aggregates. Of the uricase thus pooled, at least90% may be in the tetrameric form; the undesirable aggregates may thusconstitute as little as about 10%, 5%, 2%, or less, of the totalisolated uncase.

[0054] The results presented herein indicate that, even when extensivelyPEGylated, forms of PBC uricase larger than the tetramer are highlyimmunogenic in mice (FIG. 12). Furthermore, in mice that had beeninjected once with PEG conjugates of uncase aggregates, the uricolyticactivity in subsequent injections of either PEGylated tetramers orPEGylated aggregates was cleared rapidly from the circulation. Incontrast, conjugates prepared from uricase containing less than 5%aggregates could be reinjected many times without any acceleration oftheir clearance rates (FIG. 7) and without the detectable formation ofantibodies, as measured by a sensitive enzyme-linked immunoassay. Theuse of highly purified tetrameric uricase further distinguishes theimproved conjugates of the present invention from the PEG-uricasepreparations described previously. In contrast, the presence of asignificant proportion (e.g., >10%) of aggregates in the uricasepreparations used by some previous investigators may have led them tocouple large numbers of strands of PEG in efforts to suppress theimmunogenicity. Consequently, the enzymatic activity of the resultantconjugates was decreased substantially. In other embodiments, thepresent invention expressly contemplates PEGylated uricase innon-tetrameric form, such as, for example, uricase dimers, so long asthe preparations of such conjugated uricase retain at least about 75% oftheir uricolytic activity and are substantially non-immunogenic.

[0055] In another embodiment of the present invention, a mutated baboonliver uricase of unexpectedly increased potency, relative to that of theunmutated enzyme, is provided. This improved primate uricase wasprepared by conventional recombinant DNA techniques. It was particularlyunexpected that the substitution of a single amino acid residue(histidine for tyrosine at position 97) in baboon uricase would resultin a substantial increase in specific enzymatic activity. When expressedin E. coli, this mutant protein was found to have at least 60% higherspecific activity than the recombinant baboon enzyme from which it wasderived.

[0056] In another embodiment, the specific activity is increased and/orthe solubility of the unPEGylated enzyme is improved by expressingtruncated variants of porcine or porcine-baboon chimeric uricases fromwhich at least the first six amino acids at the amino terminal and/or atleast the last three amino acids at the carboxyl terminal are deletedfrom the expressed proteins (see FIG. 6). Recombinant uricases with thecarboxyl-terminal truncation may have improved solubility prior toPEGylation because of the removal of the peroxisomal targeting sequence.See Miura, S, et al., (1994) Eur J Biochem 223:141-146.

[0057] The PEG-uricase conjugates of the present invention are usefulfor lowering the levels of uric acid in the body fluids and tissues ofmammals, preferably humans, and can thus be used for treatment ofelevated uric acid levels associated with conditions including gout,tophi, renal insufficiency, organ transplantation and malignant disease.PEG-uricase conjugates may be injected into a mammal having excessiveuric acid levels by any of a number of routes, including intravenous,subcutaneous, intradermal, intramuscular and intraperitoneal routes.Alternatively, they may be aerosolized and inhaled. See Patton, J S,(1996) Adv Drug Delivery Rev 19:3-36 and U.S. Pat. No. 5,458,135. Theeffective dose of PEG-uricase of the present invention will depend onthe level of uric acid and the size of the individual. In one embodimentof this aspect of the invention, PEG-uricase is administered in apharmaceutically acceptable excipient or diluent in an amount rangingfrom about 10 μg to about 1 g. In a preferred embodiment, the amountadministered is between about 100 μg and 500 mg. More preferably, theconjugated uricase is administered in an amount between 1 mg and 100 mg,such as, for example, 5 mg, 20 mg or 50 mg. Masses given for dosageamounts of the embodiments refer to the amount of protein in theconjugate.

[0058] Pharmaceutical formulations containing PEG-uricase can beprepared by conventional techniques, e.g., as described in Gennaro, A R(Ed.) (1990) Remington's Pharmaceutical Sciences, 18th Edition Easton,Pa.: Mack Publishing Co. Suitable excipients for the preparation ofinjectable solutions include, for example, phosphate buffered saline,lactated Ringer's solution, water, polyols and glycerol. Pharmaceuticalcompositions for parenteral injection comprise pharmaceuticallyacceptable sterile aqueous or non-aqueous liquids, dispersions,suspensions, or emulsions as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.These formulations may contain additional components, such as, forexample, preservatives, solubilizers, stabilizers, wetting agents,emulsifiers, buffers, antioxidants and diluents.

[0059] PEG-uricase may also be provided as controlled-releasecompositions for implantation into an individual to continually controlelevated uric acid levels in body fluids. For example, polylactic acid,polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate,gellan gum, chitosan, agarose, multilamellar liposomes and many otherconventional depot formulations comprise bioerodible or biodegradablematerials that can be formulated with biologically active compositions.These materials, when implanted or injected, gradually break down andrelease the active material to the surrounding tissue. For example, onemethod of encapsulating PEG-uricase comprises the method disclosed inU.S. Pat. No. 5,653,974, which is hereby incorporated by reference. Theuse of bioerodible, biodegradable and other depot formulations isexpressly contemplated in the present invention. The use of infusionpumps and matrix entrapment systems for delivery of PEG-uricase is alsowithin the scope of the present invention. PEG-uricase may alsoadvantageously be enclosed in micelles or liposomes. Liposomeencapsulation technology is well known in the art. See, e.g., Lasic, D,et al., (Eds.) (1995) Stealth Liposomes. Boca Raton, Fla.: CRC Press.

[0060] The PEG-uricase pharmaceutical compositions of the invention willdecrease the need for hemodialysis in patients at high risk ofurate-induced renal failure, e.g., organ transplant recipients (seeVenkataseshan, V S, et al., (1990) Nephron 56:317-321) and patients withsome malignant diseases. In patients with large accumulations ofcrystalline urate (tophi), such pharmaceutical compositions will improvethe quality of life more rapidly than currently available treatments.

[0061] The following examples, which are not to be construed as limitingthe invention in any way, illustrate the various aspects disclosedabove. These examples describe PEG-uricases prepared by couplingactivated (i.e., electrophilic) PEG derivatives of several sizes andcompositions with naturally occurring porcine, fungal or bacterialuricases, or with recombinant soybean, porcine or pig-baboon chimericuricases. Results of activity, solubility, stability, pharmacokinetic,pharmacodynamic and immunological studies are included. The data inFIGS. 8-11 provide evidence of the ability of PEG-modified PBC uricaseof this invention to correct hyperuricemia and hyperuricosuria and topreserve renal structure and function in an animal model in whichhyperuricemia and hyperuricosuria occur and cause serious renal damage.Wu, X, et al., (1994) Proc Natl Acad Sci USA 91:742-746. These examplesprovide guidance to one with ordinary skill in the art for producingsubstantially non-immunogenic conjugates of uricase that retain at leastabout 75% of the uricolytic activity of the unmodified enzyme.

EXAMPLE 1 Purification of the Tetrameric Form of Uricase

[0062] The tetrameric form of uricase (molecular weight ca. 140 kDa) waspurified from a solution of porcine liver uricase by preparativesize-exclusion or ion-exchange chromatography, followed by analyticalsize-exclusion chromatography. Porcine liver uricase was obtained fromSigma-Aldrich, St. Louis, Mo., catalog No. U2350 or U3377; or BoehringerMannheim, Indianapolis, Ind.

[0063] Preparative and analytical size—exclusion chromatography wereperformed at pH 10-10.5, preferably 10.2, in 10 mM sodium carbonatebuffer containing 0.1 M NaCl on Superdex 200 columns that had beenpreviously calibrated with proteins of known molecular weight. Superdexwas obtained from Amersham Pharmacia, Piscataway, N.J. Any buffer may beused that is capable of maintaining the desired pH and that iscompatible with the chemistry to be used for subsequent PEG coupling.Such buffers are well known in the art. The ultraviolet absorbance ofthe eluate from the preparative column was monitored at 280 nm, anduricase-containing portions of the eluate corresponding to the molecularweight of the desired tetrameric form, but free of higher molecularweight species, were collected for use in synthesizing substantiallynon-immunogenic PEG-uricase as described in Example 2. Alternatively,tetrameric forms of uricase can be isolated using other size-exclusionmedia such as, for example, Superose 12 (Amersham Pharmacia) or anyother medium that is compatible with mildly alkaline solutions and thathas an appropriate size fractionation range. Such media are readilyavailable and are well known in the art.

[0064] Ion-exchange chromatography was performed at pH 10-10.5,preferably 10.2, on Mono Q columns (Amersham Pharmacia, Piscataway,N.J.) that had been equilibrated with 0.1 M sodium carbonate buffer. Anybuffer that is compatible with the chemistry of PEG coupling and that iscapable of maintaining the desired pH may be used at sufficiently lowionic strength to permit the adsorption of uricase to the column. Suchbuffers are well known in the art. The ultraviolet absorbance of theeluate was monitored at 280 nm during elution of the uricase from theion-exchange resin by increasing the ionic strength of the appliedbuffer solution, e.g. by a linear gradient of 0 to 0.5 M NaCl in thesodium carbonate buffer. Size-exclusion HPLC was then used to identifythe fractions of the eluate containing the desired tetrameric form ofuricase, without detectable aggregates, for the synthesis ofsubstantially non-immunogenic PEG-uricase. Alternatively, the tetramericform of uricase can be isolated using other ion-exchange media, such asQ-Sepharose (Amersham Pharmacia) or any other medium that is compatiblewith mildly alkaline solutions. Such media are readily available and arewell known in the art.

[0065] Uricase activity was assayed using a modification of standardmethods. See, e.g., Fridovich (1965); Nishimura, et al., (1979).Solutions of uric acid were prepared fresh daily in 50 mM sodium boratebuffer, pH 9.2, to provide final concentrations in the assay of 6-150μM. Uricase preparations were diluted in this borate buffer containingbovine serum albumin (Sigma-Aldrich, St. Louis, Mo., catalog No.A-7030), so that the final concentration of albumin in the assay was 0.1mg/mL. After mixing various dilutions of the enzyme with the substratein the wells of a microtiter plate in a microplate reader, the rate ofdisappearance of uric acid at 25° C. was monitored at 292 nm every 4seconds for 3 minutes. From samples in which between 10% and 40% of thesubstrate was consumed within 3 minutes, at least 20 data points wereused to calculate the maximal rate of decrease in the absorbance perminute. One international unit (IU) of uricase activity is defined asthe amount of enzyme that consumes one micromole of uric acid perminute; specific activities are expressed as IU/mg protein. Some of thedata for relative uricase activities in FIGS. 1A-5B were obtained using100 μM uric acid in the assay. Other results for the velocity at 100 μMuric acid (V₁₀₀) were calculated from the values of the Michaelisconstant (K_(M)) and the maximal velocity (V_(max)) for the respectiveenzyme preparations, using the formula:

V ₁₀₀=100×V _(max)/(K _(M)+100)

[0066] where K_(M) is expressed in micromolar units.

EXAMPLE 2 PEG Coupling to Tetrameric Porcine Uricase

[0067] To a solution of tetrameric uricase in 0.1 M sodium carbonatebuffer, pH 10.2, 10-200 moles of an activated derivative ofmonomethoxyPEG, e.g., the 4-nitrophenyl carbonate (NPC-PEG), of varioussizes (5 kDa to 30 kDa) were added for each mole of uricase subunit(molecular weight 35 kDa). These and other suitable activated PEGs areavailable from Shearwater Polymers. Instructions for coupling these PEGsto proteins are given in the catalog of Shearwater Polymers, on theInternet at www.swpolymers.com, and in J M Harris, et al., (Eds.) (1997)Poly(ethylene glycol) Chemistry and Biological Applications. ACSSymposium Series 680, Washington, D.C.: American Chemical Society. Thecoupling reaction was allowed to proceed at 0-8° C. until the extent ofPEG coupling no longer changed significantly with time. Unreacted PEGwas then removed from the reaction product by chromatography and/orultrafiltration.

[0068] The number of strands of PEG coupled per subunit of uricase wasdetermined by an adaptation of the methods described by Kunitani, M, etal., (1991) J Chromatogr 588:125-137; Saifer, et al., (1997) andSherman, et al., (1997). Briefly, aliquots of the PEGylation reactionmixtures or fractions from the preparative ion-exchange orsize-exclusion columns were characterized by analytical size-exclusionHPLC on a TSK 5,000 PW_(XL) column at room temperature in 10 mM sodiumcarbonate buffer, pH 10.2, containing 0.1 M NaCl. The HPLC column wasobtained from TosoHaas, Montgomeryville, Pa. Proteins and PEGs weremonitored by ultraviolet absorbance and refractive index detectors. Theamount of protein in the conjugate was calculated from the ultravioletabsorbance relative to that of the appropriate unmodified uricasestandard. The amount of PEG in the conjugate was then calculated fromthe area of the refractive index peak, corrected for the contribution ofthe protein to refractive index, relative to the area of the refractiveindex peak of the appropriate PEG standard.

[0069]FIG. 2A shows the retention of activity by PEGylated porcine liveruricase as a function of the number of strands of PEG coupled persubunit. Data of the present inventors (▴,□) are compared with those ofChen, et al., (1981). The data point within a large circle denotes aconjugate reported to be non-immunoreactive by Chen, et al., (1981). Asshown in FIG. 2A, conjugates of tetrameric porcine uricase with up to 6strands of 30 kDa PEG per subunit or up to 7 strands of 5 kDa PEG persubunit retained at least 75% of the activity of the unmodified enzyme.The apparent increase in specific activity with an increasing number ofstrands of 5 kDa or 30 kDa PEG (up to about 4 strands per subunit) mayreflect the relative insolubility or instability of the unmodifiedenzyme compared to the conjugates. As shown in FIG. 2B, conjugates ofporcine uncase with an average of more than 3 strands of 30 kDa PEG persubunit contain a greater mass of PEG than was found sufficient topreclude immunoreactivity by Chen, et al., (1981).

EXAMPLE 3 Properties of PEG Conjugates of Tetrameric Recombinant PBCUricase

[0070] Recombinant pig-baboon chimeric (PBC) uricase cDNA was subclonedinto the pET3d expression vector (Novagen, Madison, Wis.) and theresultant plasmid construct was transformed into and expressed in astrain of Escherichia coli BL21(DE3)pLysS (Novagen). These procedureswere carried out using methods well known in the art of molecularbiology. See Erlich (1989); Sambrook, et al., (1989); Ausubel, F, etal., (Eds.), (1997) Short Protocols in Molecular Biology. New York: JohnWiley & Sons.

[0071]FIG. 6 shows the deduced amino acid sequence of PBC uricase (aminoacids 1-225 of SEQ ID NO: 1 and amino acids 226-304 of SEQ ID NO: 2),compared with the porcine (SEQ ID NO: 1) and baboon (SEQ ID NO: 2)sequences. Residues in the baboon sequence that differ from those in theporcine sequence are shown in bold type. The porcine and baboonsequences were first determined by Wu, et al., (1989) and were confirmedby the present inventors. SEQ ID NO. 1 is identical to Accession Numberp16164 of GenBank, except for the absence of the initial methionylresidue in the GenBank sequence. SEQ ID NO. 2 is identical to AccessionNumber p25689 of GenBank, except for the absence of the initialmethionyl residue and a change from histidine to threonine at residue153 in the GenBank sequence (residue 154 in FIG. 6).

[0072] The tetrameric form of PBC uricase was isolated and coupled toPEGs of various molecular weights as described in Examples 1 and 2.Conjugates prepared with 5 kDa, 10 kDa, 19 kDa or 30 kDa PEG containedup to 10 strands of PEG per subunit. Those prepared with PEGs of atleast 10 kDa retained more than 95% of the initial specific activity ofthe recombinant uricase (FIGS. 3A-3B).

[0073] The following properties of a conjugate of tetrameric PBC uricasewith approximately 6 strands of 10 kDa PEG per subunit are illustratedin the indicated figures: the lack of immunogenicity (FIG. 7) and theefficacy in uricase-deficient mice in 1) correcting hyperuricemia andhyperuricosuria (FIG. 8); 2) decreasing the severity of aurine-concentrating defect (FIG. 9), and 3) decreasing the severity ofnephrogenic diabetes insipidus (FIG. 10). In addition, this PEG-uricasedecreased the severity of uric acid-related renal damage, as visualizedby magnetic resonance microscopy (FIG. 11).

[0074]FIG. 7 shows the activity of PBC uncase in mouse serum 24 h aftereach of four or five intraperitoneal injections of PEG-uricase, relativeto the value 24 h after the first injection. PEG conjugates wereprepared from three different preparations of PBC uncase using twodifferent techniques for PEG activation. One preparation () was testedin uricase-deficient (uox −/−) mice; the other two (Δ,▪) were tested innormal BALB/c mice. The most immunoreactive preparation (Δ) was preparedfrom purified PBC uricase containing an unknown quantity of uricaseaggregates coupled to an average of 7 strands of 5 kDa PEG per subunit,using the succinimidyl carbonate derivative of PEG (SC-PEG). Zalipsky,U.S. Pat. No. 5,612,460, hereby incorporated by reference. Themoderately immunoreactive preparation (▪) was prepared by coupling a PBCuricase preparation containing 11% aggregates to an average of 2 strandsof 19 kDa PEG per subunit, using a 4-nitrophenyl carbonate derivative ofPEG (NPC-PEG). Sherman, et al., (1997). The least immunoreactiveconjugate () was prepared by coupling an average of 6 strands of 10 kDaNPC-PEG per subunit to a preparation of PBC uricase containing <5%aggregated uricase.

[0075]FIG. 8 shows the inverse relationship between the concentrationsof uric acid in the serum and urine and the activity of injectedPEG-uricase in the serum of a uricase-deficient (uox −/−) mouse.Injections at zero time and after 72 h contained 0.43 IU of PBC uricaseconjugated to an average of 6 strands of 10 kDa PEG per enzyme subunit.

[0076]FIG. 9 shows that treatment of uricase-deficient mice withPEG-modified PBC uricase decreased the severity of a urine-concentratingdefect. The mean and standard deviation of data for urine osmolality areshown for two mice containing one copy of the normal murine uricase gene(uox +/−), six untreated homozygous uricase-deficient mice (uox −/−) andsix homozygous uricase-deficient mice that were injected ten timesbetween the third and 72nd day of life with either 95 or 190 mIU ofPEG-uricase. Mice of each genetic background either had received waterad libitum (solid bars) or had been deprived of water for 12 h (hatchedbars) prior to collection of their urine.

[0077]FIG. 10 shows that treatment of uricase-deficient mice withPEG-modified PBC uricase decreased the severity of nephrogenic diabetesinsipidus, characterized by abnormally high consumption of water andabnormally high urine output. The genetic backgrounds of the mice andtreatment protocol were the same as in FIG. 9. The mean and standarddeviation of the daily water consumption (solid bars) and urine output(hatched bars) are shown for three groups of six mice.

[0078]FIG. 11 shows that treatment of uricase-deficient mice withPEG-modified PBC uricase decreased the severity of uric acid-inducednephropathy, as visualized by magnetic resonance microscopy. The geneticbackgrounds of the three groups of mice and the treatment protocol werethe same as in FIGS. 9 and 10. Magnetic resonance microscopy wasperformed at the Center for in vivo Microscopy, Duke University MedicalCenter, Durham, N.C.

[0079] In addition to the results summarized in FIGS. 8-11, it wasdemonstrated that the uric acid levels in the urine of alluricase-deficient mice decreased dramatically after treatment withPEG-modified PBC uricase. Finally, FIG. 12 shows that, unlike thePEG-modified tetrameric form of PBC uricase, the octameric form(molecular weight=280 kDa), even when extensively PEGylated, isimmunogenic in mice. This property is reflected in the acceleratedclearance of the PEG-modified octamer within 5 days after a singleintraperitoneal injection. The same mice were re-injected with the samedose of the same PEG-uricase preparations on days 8 and 15. Twenty-fourhours after the second and third injections, uricolytic activity wasundetectable in the sera of mice injected with the PEGylated octamer,but was readily detected in the sera of those injected with thePEGylated tetramer. These findings, in combination with the acceleratedclearance of the PEGylated octamer observed after the first injection(FIG. 12), support the utility of removing all forms of uricase largerthan the tetramer prior to PEGylation of the enzyme.

EXAMPLE 4 PEG Conjugation of Uricase from Candida utilis

[0080] Uricase from Candida utilis was obtained from eitherSigma-Aldrich (St. Louis, Mo.; catalog No. U1878) or WorthingtonBiochemical Corporation (Freehold, N.J.; catalog No. URYW). Proceedingas described in Examples 1 and 2, the tetrameric form was isolated andPEG conjugates were synthesized with 5 kDa, 10 kDa or 30 kDa PEG (FIGS.1A-1B). FIG. 1A shows the retention of activity by PEGylated uricasefrom Candida utilis as a function of the number of strands of PEGcoupled per subunit. Data of the present inventors (▴, , □) arecompared with those of Nishimura, et al., (1979); Nishimura, et al.,(1981); Chen, et al., (1981); Davis, et al., (1981); Tsuji, et al.,(1985); Yasuda, et al., (1990), and Fujita, et al., (1991). Data pointswithin large circles denote conjugates reported to be non-antigenic byNishimura, et al., (1979 or 1981) or non-immunoreactive by Chen, et al.,(1981).

[0081]FIG. 1B shows the retention of activity by PEGylated uricase fromCandida utilis as a function of the total mass of PEG coupled persubunit. Data of the present inventors (▴, , □) are compared with thoseof the same reports as in FIG. 1A. Data points within large circles havethe same meaning as in FIG. 1A.

[0082] As shown in FIGS. 1A and 1B, conjugates with an average of up to6 strands of 5 kDa or 30 kDa PEG or 9 strands of 10 kDa PEG per subunitretained at least 75% of the activity of the unmodified enzyme. Theapparent increase in specific activity as an increasing number ofstrands of 30 kDa PEG is attached (up to 5 or 6 strands per subunit) mayreflect the relative insolubility or instability of the unmodifiedenzyme compared to the conjugates.

EXAMPLE 5 PEG Conjugation of Uricase from Aspergillus flavus

[0083] Uricase from Aspergillus flavus was obtained from Sanofi Winthrop(Gentilly Cedex, France). Proceeding as described in Example 2,conjugates with PEGs of various molecular weights were synthesized(FIGS. 4A-4B). Conjugates prepared by coupling the enzyme from A. flavuswith an average of up to 12 strands of 5 kDa PEG or up to 7 strands of30 kDa PEG per subunit retained at least 75% of the initial specificactivity of this fungal uricase.

EXAMPLE 6 PEG conjugation of Soybean Uricase

[0084] Recombinant uricase from soybean root nodule (also called nodulin35) was prepared and purified as described by Kahn and Tipton (Kahn, K,et al., (1997) Biochemistry 36:4731-4738), and was provided by Dr.Tipton (University of Missouri, Columbia, Mo.). Proceeding as describedin Examples 1 and 2, the tetrameric form was isolated and conjugateswere prepared with PEGs of various molecular weights (FIGS. 5A-5B). Incontrast to uricase from Candida utilis (FIG. 1A), porcine uricase (FIG.2A), pig-baboon chimeric uricase (FIG. 3A) and uricase from Aspergillusflavus (FIG. 4A), the soybean enzyme tolerated coupling of onlyapproximately 2 strands of 5 kDa or 30 kDa PEG per subunit withretention of at least 75% of the initial uricolytic activity.

EXAMPLE 7 PEG Conjugation of Uricase from Arthrobacter globiformis

[0085] Uricase from Arthrobacter globiformis was obtained fromSigma-Aldrich (catalog No. U7128). See Japanese Patent 9-154581.Proceeding as described in Examples 1 and 2, the tetrameric form wasisolated and conjugates with 5 kDa and 30 kDa PEG were prepared. Whileconjugates with an average of more than 3 strands of 5 kDa PEG persubunit retained less than 60% of the initial specific activity,conjugates with an average of approximately 2 strands of 30 kDa PEG persubunit retained at least 85% of the initial specific activity.

EXAMPLE 8 PEG Conjugation of Amino-Truncated Porcine and PBC Uricases

[0086] Recombinant porcine and PBC uricases from which the first sixamino acids at the amino terminal are deleted are expressed in andpurified from E. coli by standard techniques, as described in Example 3.Proceeding as described in Examples 1 and 2, PEG conjugates of theamino-truncated uricases are synthesized to produce substantiallynon-immunogenic conjugates that retain at least 75% of the initialspecific activity.

EXAMPLE 9 PEG Conjugation of Porcine and PBC Uricases Truncated at theCarboxyl Terminal or Both the Amino and Carboxyl Terminals

[0087] Recombinant porcine and PBC uricases from which the last threeamino acids at the carboxyl terminal are deleted are expressed in andpurified from E. coli by standard techniques, as described in Example 3.This carboxyl-terminal deletion may enhance the solubility of theunmodified enzymes, since it removes the peroxisomal-targeting signal.See Miura, et al., (1994). Proceeding as described in Examples 1 and 2,PEG conjugates of the carboxyl-truncated uricases are synthesized toproduce substantially non-immunogenic conjugates that retain at least75% of the initial specific activity. The sequence of recombinant PBCuricase truncated by six residues at the amino terminal and by threeresidues at the carboxyl terminal (PBC-NT-CT) is shown in FIG. 6. Thisuricase is expressed, purified and PEGylated as described in Examples 1,2 and 3 to produce substantially non-immunogenic conjugates that retainat least 75% of the initial specific activity.

EXAMPLE 10 PEG Conjugation of Porcine Uricase Mutants Containing anIncreased Number of PEG Attachment Sites

[0088] Recombinant porcine uricases are prepared as described in Example3, in which the potential number of sites of PEG attachment is increasedby replacing one or more arginine residues with lysine. See Hershfield,M S, et al., (1991) Proc Natl Acad Sci USA 88:7185-7189. The amino acidsequence of one example of such a mutant (PKS uricase), in which thearginine at residue 291 is replaced by lysine and the threonine atresidue 301 is replaced by serine, is shown in FIG. 6. Proceeding asdescribed in Examples 1 and 2, PEG is conjugated to this uricase toproduce substantially non-immunogenic conjugates that retain at least75% of the initial specific activity of the recombinant uricase.

EXAMPLE 11 PEG Conjugation of a Recombinant Baboon Uricase Mutant

[0089] Using standard methods of molecular biology, as in Example 3,recombinant baboon uricase is constructed having an amino acidsubstitution (histidine for tyrosine) at position 97 (see baboonsequence in FIG. 6). Proceeding as described in Examples 1 and 2, PEGconjugates of the tetrameric form of the recombinant baboon uricasemutant are synthesized to produce conjugates of substantially reducedimmunogenicity that retain at least 75% of the initial specific activityof the recombinant uricase.

EXAMPLE 12 Immunogenicity of PEG Conjugates from Candida utilis,Aspergillus flavus, and Arthrobacter globiformis

[0090] Uricase from Candida utilis, Aspergillus flavus, and Arthrobacterglobiformis are obtained as described in Examples 4, 5, and 7,respectively. Proceeding as described in Examples 1 and 2, PEGconjugates are synthesized with 5 kDa, 10 kDa, 20 kDa or 30 kDa PEG. Theimmunogenicity of these conjugates is substantially reduced oreliminated.

1 3 1 304 PRT Sus scrofa 1 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys AsnAsp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met IleLys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser Ile Lys GluVal Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr LeuHis Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr Asp Thr Ile Lys Asn ThrVal Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly Ile Lys Ser Ile Glu ThrPhe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His ValIle Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg PheGlu Lys Asn Gly Val Lys His Val 115 120 125 His Ala Phe Ile Tyr Thr ProThr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Ile Arg Asn Gly ProPro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155 160 Lys Val Leu LysThr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165 170 175 Gln Phe ThrThr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln 180 185 190 Val TyrCys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 AlaThr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly 210 215 220Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met SerLys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu AspAsn Pro 275 280 285 Tyr Gly Arg Ile Thr Gly Thr Val Lys Arg Lys Leu ThrSer Arg Leu 290 295 300 2 304 PRT Papio hamadryas 2 Met Ala Asp Tyr HisAsn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe 1 5 10 15 Val Arg Thr GlyTyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly LysTyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu SerSer Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Ile Ile Pro ThrAsp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys 65 70 75 80 Phe Lys GlyIle Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu 85 90 95 Tyr Phe LeuSer Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu GluIle Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115 120 125 HisAla Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp165 170 175 Gln Phe Thr Thr Lys Pro Glu Val Lys Asp Arg Cys Phe Ala ThrGln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val AspPhe Glu 195 200 205 Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu LysPhe Ala Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val GlnLys Thr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Ser Leu Ser Arg ValPro Glu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His TyrPhe Asn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu GluVal Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly ThrVal Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 3 304 PRT Mutantcombination of Sus scrofa & Papio hamadryas 3 Met Ala His Tyr Arg AsnAsp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly TyrGly Lys Asp Met Ile Lys Val Leu His Ile Gln 20 25 30 Arg Asp Gly Lys TyrHis Ser Ile Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser SerLys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val Ile Pro Thr AspThr Ile Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly IleLys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu 85 90 95 His Phe Leu SerSer Phe Lys His Val Ile Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu ValPro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His AlaPhe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 GlnIle Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu 145 150 155160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp 165170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp PheGlu 195 200 205 Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys PheAla Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln LysThr Leu Tyr 225 230 235 240 Asp Ile Gln Val Leu Thr Leu Gly Gln Val ProGlu Ile Glu Asp Met 245 250 255 Glu Ile Ser Leu Pro Asn Ile His Tyr LeuAsn Ile Asp Met Ser Lys 260 265 270 Met Gly Leu Ile Asn Lys Glu Glu ValLeu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys Ile Thr Gly Thr ValLys Arg Lys Leu Ser Ser Arg Leu 290 295 300

What is claimed is:
 1. A conjugate of uricase that retains at leastabout 75% of the uricolytic activity of unconjugated uricase and issubstantially non-immunogenic, comprising a purified uricase comprisingsubunits in which each subunit of the uricase is covalently linked to anaverage of 2 to 10 strands of PEG, wherein each molecule of PEG has amolecular weight between about 5 kDa and 100 kDa.
 2. The conjugate ofclaim 1, wherein the uricase is mammalian uricase.
 3. The conjugate ofclaim 2, wherein the uricase is porcine liver, bovine liver or ovineliver uricase.
 4. The conjugate of claim 1, wherein the uricase isrecombinant.
 5. The conjugate of claim 4, wherein the uricase hassubstantially the sequence of porcine, bovine, ovine or baboon liveruricase.
 6. The conjugate of claim 4, wherein the uricase is chimeric.7. The conjugate of claim 6, wherein the chimeric uricase containsportions of porcine liver and baboon liver uricase.
 8. The conjugate ofclaim 7, wherein the chimeric uricase is PBC uricase.
 9. The conjugateof claim 7, wherein the chimeric uricase is PKS uricase.
 10. Theconjugate of claim 4, wherein the uricase has substantially the sequenceof baboon liver uricase in which tyrosine 97 has been replaced byhistidine.
 11. The conjugate of claim 4, wherein the uricase comprisesan amino terminal and a carboxyl terminal, and wherein the uricase istruncated at one terminal or both terminals.
 12. The conjugate of claim1, wherein the uricase is a fungal or microbial uricase.
 13. Theconjugate of claim 12, wherein the fungal or microbial uricase isisolated from Aspergillus flavus, Arthrobacter globiformis or Candidautilis, or is a recombinant enzyme having substantially the sequence ofone of those uricases.
 14. The conjugate of claim 1, wherein the uricaseis an invertebrate uricase.
 15. The conjugate of claim 14, wherein theinvertebrate uricase is isolated from Drosophila melanogaster orDrosophila pseudoobscura, or is a recombinant enzyme havingsubstantially the sequence of one of those uricases.
 16. The conjugateof claim 1, wherein the uricase is a plant uricase.
 17. The conjugate ofclaim 16, wherein the plant uricase is isolated from root nodules ofGlycine max or is a recombinant enzyme having substantially the sequenceof that uricase.
 18. The conjugate of claim 1, wherein the PEG has anaverage molecular weight between about 10 kDa and 60 kDa.
 19. Theconjugate of claim 18, wherein the PEG has an average molecular weightbetween about 20 kDa and 40 kDa.
 20. The conjugate of claim 1, whereinthe average number of covalently coupled strands of PEG is 3 to 8strands per uricase subunit.
 21. The conjugate of claim 20, wherein theaverage number of covalently coupled strands of PEG is 4 to 6 strandsper uricase subunit.
 22. The conjugate of claim 1, wherein the uricaseis tetrameric.
 23. The conjugate of claim 1, wherein the strands of PEGare covalently coupled to uricase via linkages selected from the groupconsisting of urethane linkages, secondary amine linkages, and amidelinkages.
 24. The conjugate of claim 1, wherein the PEG is linear. 25.The conjugate of claim 1, wherein the PEG is branched.
 26. Apharmaceutical composition for lowering uric acid levels in a body fluidor tissue, comprising the conjugate of claim 1 and a pharmaceuticallyacceptable carrier.
 27. The pharmaceutical composition of claim 26,wherein said composition is stabilized by lyophilization and dissolvespromptly upon reconstitution to provide solutions suitable forparenteral administration.
 28. A method for lowering elevated uric acidlevels in a body fluid or tissue of a mammal, comprising the step ofadministering to said mammal an effective uric acid-lowering amount ofPEG-uricase, said PEG-uricase comprising a purified uricase comprisingat least two subunits in which each subunit is covalently linked to anaverage of 2 to 10 strands of PEG, wherein each molecule of PEG has amolecular weight between about 5 kDa and 100 kDa, in a pharmaceuticallyacceptable carrier.
 29. The method of claim 28, wherein said mammal is ahuman.
 30. The method of claim 28, wherein the administering step isselected from the group consisting of injections by intravenous,intradermal, subcutaneous, intramuscular and intraperitoneal routes orinhalation of an aerosolized formulation.
 31. The method of claim 28,wherein said elevated uric acid levels are associated with a conditionselected from the group consisting of gout, tophi, renal insufficiency,organ transplantation and malignant disease.
 32. The method of claim 28,wherein the PEG is linear.
 33. The method of claim 28, wherein the PEGis branched.
 34. A method for isolating a tetrameric form of uricasefrom a solution of uricase, said solution comprising tetrameric uricaseand uricase aggregates, comprising the steps of: applying said solutionto at least one separation column at a pH between about 9 and 10.5; andrecovering from said column one or more fractions that contain isolatedtetrameric uricase, wherein said one or more fractions are substantiallyfree of uricase aggregates.
 35. The method of claim 34, wherein saidsolution of said uricase is applied to said column at a pH of 10.2. 36.The method of claim 34, wherein said separation is based on a propertyselected from the group consisting of ion exchange and size exclusion.37. The method of claim 34, further comprising the step of analyzingsaid fractions to determine at least one property selected from thegroup consisting of presence of said tetrameric uricase and absence ofuricase aggregates.
 38. The method of claim 37, wherein said analyzingstep comprises at least one analysis selected from the group consistingof chromatography, centrifugation, light scattering and electrophoresis.39. The method of claim 38, wherein said chromatography is highperformance liquid chromatography.
 40. The method of claim 34, whereinsaid isolated tetrameric uricase contains less than about 10% uricaseaggregates.
 41. An isolated tetrameric uricase produced by the method ofclaim
 34. 30. The method of claim 28, wherein the administering step isselected from the group consisting of injections by intravenous,intradermal, subcutaneous, intramuscular and intraperitoneal routes orinhalation of an aerosolized formulation.
 31. The method of claim 28,wherein said elevated uric acid levels are associated with a conditionselected from the group consisting of gout, tophi, renal insufficiency,organ transplantation and malignant disease.
 32. The method of claim 28,wherein the PEG is linear.
 33. The method of claim 28, wherein the PEGis branched.
 34. A method for isolating a tetrameric form of uricasefrom a solution of uricase, said solution comprising tetrameric uricaseand uricase aggregates, comprising the steps of: applying said solutionto at least one separation column at a pH between about 9 and 10.5; andrecovering from said column one or more fractions that contain isolatedtetrameric uricase, wherein said one or more fractions are substantiallyfree of uricase aggregates.
 35. The method of claim 34, wherein saidsolution of said uricase is applied to said column at a pH of 10.2. 36.The method of claim 34, wherein said separation is based on a propertyselected from the group consisting of ion exchange and size exclusion.37. The method of claim 34, further comprising the step of analyzingsaid fractions to determine at least one property selected from thegroup consisting of presence of said tetrameric uricase and absence ofuricase aggregates.
 38. The method of claim 37, wherein said analyzingstep comprises at least one analysis selected from the group consistingof chromatography, centrifugation, light scattering and electrophoresis.39. The method of claim 38, wherein said chromatography is highperformance liquid chromatography.
 40. The method of claim 34, whereinsaid isolated tetrameric uricase contains less than about 10% uricaseaggregates.
 41. An isolated tetrameric uricase produced by the method ofclaim 34.